(2S)-2-amino-3-(4-{[bis(sodiooxy)phosphoryl]oxy}phenyl)propanoic acid

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014-07-07 to 2014-11-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

(2S)-2-amino-3-(4-{[bis(sodiooxy)phosphoryl]oxy}phenyl)propanoic acid

EC Number:

860-352-3

Cas Number:

1610350-91-8

Molecular formula:

C9H10NO6PNa2

IUPAC Name:

(2S)-2-amino-3-(4-{[bis(sodiooxy)phosphoryl]oxy}phenyl)propanoic acid

Test material form:

solid: particulate/powder

Method

Target gene:

- HIS operon (Salmonella typhimurium strains)
- TRP operon (Escherichia coli WP2 uvrA)

The strains are constructed to differentiate between base pair (E.coli WP2 uvrA, TA1535, TA100, TA102) and frameshift (TA1537, TA98) mutations.

Species / strain

Species / strain 1

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:

- source of S9: Aroclor-induced male Wistar rats

- method of preparation of S9 mix: Male Wistar rats were given a single intraperitoneal injection of Aroclor 1254 (500 mg/kg body weight) dissolved in Miglyol 812 oil. On day 5 to 7, they were sacrificed, the livers were removed and collected in ice-cooled sterilized beakers containing 0.15 M KCl. After homogenization the preparation was transferred to sterilized steel centrifuge tubes and spun al 9000 x g for 10 minutes al about 4°C and the supernatant fluid was decanted and transferred into sterilized and prccooled plastic tubes. The S9 was then frozen and stored in liquid nitrogen at -196°C.

- concentration or volume of S9 mix and S9 in the final culture medium:
S9: 0.1 mL (1st series) and 0.3 mL (2nd series)

- quality controls of S9: Every S9-batch is tested for its metabolic activity by the use of specific substrates, requiring different enzymes of the P450-isoenzyme family. The mutagenicity of 2-aminoanthracene, benzo[a]pyrene, and 3-methylcholanthrene is thus determined once for every S9-batch.

Test concentrations with justification for top dose:

- 1st series: 5, 15.8, 50, 158, 500, 1580, 5000 µg/plate
- 2nd series: 50, 158, 500, 1580, 2810, 5000 µg/plate

A maximum concentration of 5000 µg/plate was selected as the maximum recommended concentration according to current regulatory guidelines (OECD, 1997).

Vehicle / solvent:

- Vehicles/solvents used: DMSO (cumene hydroperoxide, 2-aminoanthracene, benzo[a]pyrene, 4-nitroquinoline-N-oxide), ethanol (9-aminoacridine), ultrapure water (daunomycin, sodium azide, test item)

- Justification for choice of solvent/vehicle: Preliminary solubility data indicated that the test item was water soluble. Based on these preliminary data, ultrapure water was selected as vehicle for the current experiment.

- Justification for percentage of solvent in the final culture medium: Since on the one hand organic solvents may have diverse effects on e.g. gene regulation and, on the other hand, high amounts of solvent will dilute the top agar, the maximum amount of solvent was limited to 100 µL per plate for water and DMSO. For ethanol, acetone and other organic solvents, 10 µL per plate was not exceeded.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

ultrapure water

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9, tested with TA 98, TA 100, and TA 1537

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

ultrapure water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: daunomycin

Remarks:

without S9, tested with TA 98, TA 100, and TA 1537

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9, tested with TA 98, TA 100, and TA 1537

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9, tested with TA 98, TA 100, and TA 1537

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with S9, tested with TA 98, TA 100, TA 102 and TA 1537

Controls 6

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S9, tested with TA 98, TA 100, TA 102, TA 1537 and TA 1535

Controls 7

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cumene hydroperoxide

Remarks:

without S9, tested with TA 98, TA 100, and TA 1537

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 2-3 days at 36-38°C


METHODS FOR MEASUREMENTS OF GENOTOXICIY
mean numbers of revertants

Evaluation criteria:

The assessment of test material-induced effects is dependent on the number of spontaneous revertants of each bacterial strain (solvent controls) and the increase in the number of revertants at the test material concentration which shows the highest number of colonies. Criteria, which are based upon the historical controls of the laboratory and statistical considerations are shown in table 1.

Statistics:

n.a.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 6

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS

- Precipitation and time of the determination: The test material did not precipitate

STUDY RESULTS
- Concurrent vehicle negative and positive control data: See tables in "Attached background material"

Ames test:
- Signs of toxicity: The test item produced no signs of toxicity to the bacteria up to the highest concentration applied.
- Mean number of revertant colonies per plate and standard deviation: See tables in "Attached background material"

Applicant's summary and conclusion

Conclusions:

With and without addition of S9 mix as the external metabolizing system,the test item was not mutagenic in this in vitro gene mutation study in bacteria.

Executive summary:

The test item was examined for its mutagenic activity in two series of in vitro microbial assays em­ploying Salmonella typhimurium TA98, TA 100, TA 102, TA 1535 and TA 1537,and Escherichia coli WP2 uvrA as indicator organisms. The test material was tested directly and after metabolic activation by liver S9 mix obtained from rats pretreated with Aroclor 1254. Precipitation of the test material on the agar plates was not occurred. Toxicity to the bacteria was not observed.

 

The validity of the mutation assay can be assessed by the results obtained for the negative and positive controls. The negative control mutant frequencies were all in the regular range. Slight deviations from those values, if they occur, are accepted if they appear in only one test series and, furthermore, all other parameters of that series are in the regular range. The strain specific positive control test materials, namely daunomycin, sodium azide, 4-nitroquinolin-N-ox­ide, 9-aminoacridine, and cumene hydroperoxide in the absence of S9 mix, yielded the expected mutant frequencies that were greatly in excess of the negative controls. The genotype of the tester strains used was thus confirmed. 2-aminoanthracene and benzo[a]pyrene, which require metabolic activation, were strongly mutagenic. This indicates that the exogenous metabolizing system used in the present investigation (S9 mix) was functioning. Thus, the demands predetermined in the study protocol and in section 4 of this report have been met in total and the study is considered valid.

 

In both series of experiments, each performed with and without the addition of rat liver S9 mix as the external metabolizing system, the test item showed no relevant increase in the number of revertants of any bacterial strain. According to the criteria for negative and positive results predetermined in the study protocol, the test item was not mutagenic under the described experimental conditions.